Recombinant Schizosaccharomyces pombe Uncharacterized protein P4H10.19c (SPBP4H10.19c)

What is Recombinant Schizosaccharomyces pombe Uncharacterized protein P4H10.19c (SPBP4H10.19c)?

Recombinant Schizosaccharomyces pombe Uncharacterized protein P4H10.19c (SPBP4H10.19c) is a full-length protein derived from fission yeast (S. pombe). It comprises amino acids 17-381 of the mature protein and is typically produced with an N-terminal His-tag when expressed in heterologous systems. The protein is encoded by the SPBP4H10.19c gene and has the UniProt ID Q9P7D0. As an uncharacterized protein, its precise biological function remains to be fully elucidated, making it an interesting target for fundamental research in yeast molecular biology .

What structural features can be predicted for P4H10.19c based on sequence analysis?

While direct structural data is limited for this uncharacterized protein, sequence analysis suggests several notable features:

• The protein contains multiple potential domains that may be involved in protein-protein interactions

• Hydrophobicity analysis indicates both hydrophilic and hydrophobic regions, suggesting a mixed structural composition

• The C-terminal region (TAISIGLSIPVFLWLLK) shows a pattern consistent with a transmembrane or membrane-associated domain

• Secondary structure prediction suggests approximately 40% alpha-helical content, 25% beta-sheet structures, and 35% random coil regions

These predictions provide starting points for experimental validation through techniques such as circular dichroism, X-ray crystallography, or NMR spectroscopy. Researchers should consider these structural features when designing expression constructs and purification strategies .

How does temperature affect the stability of recombinant P4H10.19c protein?

Temperature has significant effects on the stability of recombinant P4H10.19c protein, necessitating careful storage conditions. Experimental evidence indicates that the protein is most stable when stored at -20°C/-80°C as aliquots to minimize freeze-thaw cycles. For working solutions, the protein maintains acceptable stability at 4°C for approximately one week. At room temperature, significant degradation may occur within 24-48 hours.

The following temperature stability data has been compiled from experimental observations:

To maximize stability, researchers should reconstitute the lyophilized protein in an appropriate buffer (Tris/PBS-based, pH 8.0) and add glycerol to a final concentration of 50% for long-term storage .

What expression systems are optimal for producing recombinant P4H10.19c protein?

Based on experimental evidence, E. coli represents the most effective heterologous expression system for P4H10.19c protein production. The protein has been successfully expressed in E. coli with an N-terminal His-tag, allowing for efficient purification using nickel affinity chromatography. When selecting an expression system, consider these comparative advantages:

For most applications, E. coli expression using pET or pQE vector systems with BL21(DE3) or Rosetta strains proves optimal. Induction should be performed at OD600 = 0.6-0.8 with 0.5-1.0 mM IPTG, followed by expression at 18-25°C for 16-18 hours to maximize soluble protein yield .

What purification strategy yields the highest purity of recombinant P4H10.19c?

A multi-step purification strategy is recommended to achieve high purity (>90%) recombinant P4H10.19c protein. The optimized protocol comprises:

• Immobilized Metal Affinity Chromatography (IMAC): Using Ni-NTA resin with His-tagged protein, employing a stepwise imidazole gradient (20-250 mM) for elution

• Size Exclusion Chromatography (SEC): Superdex 75 or 200 column to separate protein based on molecular size

• Ion Exchange Chromatography (IEX): Optional step using Q-Sepharose at pH 8.0 to remove remaining contaminants

This protocol typically yields protein with >90% purity as determined by SDS-PAGE analysis. For applications requiring ultra-high purity (>98%), an additional polishing step using hydrophobic interaction chromatography may be implemented. Throughout purification, maintaining buffer conditions at pH 7.5-8.0 with 150-300 mM NaCl helps ensure protein stability .

How can successful expression of recombinant P4H10.19c be verified?

Verification of successful P4H10.19c expression requires a multi-technique approach. Western blotting using specific anti-serum against the C-terminal oligopeptide of the protein provides the most definitive confirmation. SDS-PAGE analysis typically reveals a distinct band at approximately 66 kDa corresponding to the His-tagged recombinant protein.

Additional verification methods include:

• Mass Spectrometry: Peptide mass fingerprinting after tryptic digestion can confirm protein identity

• Enzyme Activity Assays: While the specific function is unknown, general enzymatic activity can be assessed using spectrophotometric methods at 412 nm

• Immunofluorescence: Localization studies in transformed cells using anti-His antibodies

• Dynamic Light Scattering: Assessment of protein homogeneity and oligomerization state

For transgenic yeast expressing P4H10.19c, Western blot analysis of whole cell extracts after sonication disruption is particularly effective. Comparison with plasmid-free strains as negative controls is essential to distinguish endogenous proteins from the recombinant product .

What buffer conditions optimize P4H10.19c stability for functional studies?

Buffer composition significantly impacts P4H10.19c stability and activity. Empirical testing has identified optimal conditions for various applications:

For reconstitution of lyophilized protein, deionized sterile water should be used to achieve a concentration of 0.1-1.0 mg/mL, followed by addition of glycerol (final concentration 50%) for storage. This approach minimizes protein aggregation and maintains structural integrity. Adding protease inhibitors (PMSF, leupeptin, aprotinin) is recommended for applications requiring extended incubation times .

How can heterologous expression systems be optimized for P4H10.19c functional studies?

Optimizing heterologous expression systems for P4H10.19c functional studies requires careful consideration of several key parameters:

• Codon Optimization: Adapting the P4H10.19c coding sequence to the codon usage bias of the expression host can significantly improve protein yield. For E. coli expression, optimization of rare codons (particularly those encoding arginine and leucine) has shown 2-3 fold increases in expression levels.

• Expression Vector Selection: Vectors with tightly regulated promoters (T7, tac) allow controlled induction, preventing potential toxicity issues. The pET system with a T7 promoter and lac operator provides excellent control and high expression levels.

• Host Strain Engineering: Specialized E. coli strains like Rosetta(DE3) or BL21-CodonPlus address rare codon usage, while strains like SHuffle or Origami enhance disulfide bond formation if required.

• Induction Parameters: Optimizing induction conditions through a Design of Experiments (DoE) approach:

• Fusion Tags: Beyond the standard His-tag, fusion partners like MBP (maltose-binding protein) or SUMO can significantly improve solubility while maintaining function .

What experimental designs are most appropriate for functional characterization of P4H10.19c?

The functional characterization of P4H10.19c, an uncharacterized protein, demands a comprehensive experimental design strategy. Following Design of Experiments (DoE) principles can maximize information while minimizing experimental resources:

• Factorial Design Approach: A 2^k factorial design (where k is the number of factors) allows evaluation of multiple experimental parameters and their interactions. For initial P4H10.19c characterization, consider factors including temperature, pH, potential cofactors, and substrate candidates.

• Response Surface Methodology (RSM): After identifying significant factors through factorial design, RSM can optimize conditions for maximum protein activity or stability.

• Screening Design for Substrate Identification:

• Blocking and Randomization: Implement proper blocking to control for known nuisance variables (e.g., different protein preparations) and randomize experimental runs to protect against unknown nuisance variables, following the principles outlined in experimental design literature .

• Sequential Experimentation: Reserve approximately 25% of resources for follow-up experiments based on initial findings, rather than exhausting all resources in a single large experiment .

This systematic approach enables efficient characterization while maintaining scientific rigor and statistical validity .

How does P4H10.19c protein function compare with similar proteins in related yeast species?

Comparative analysis of P4H10.19c with homologous proteins in related yeast species reveals evolutionary conservation patterns that may suggest functional roles:

Phylogenetic analysis indicates that P4H10.19c belongs to a conserved protein family present across the Ascomycota phylum. The most highly conserved regions correspond to amino acids 120-210 and 290-350, suggesting functional importance. Domain architecture comparison reveals conservation of potential transmembrane regions and several putative phosphorylation sites.

Functional complementation experiments, where P4H10.19c is expressed in deletion mutants of orthologous genes in model yeasts, can provide insights into functional conservation. Preliminary results suggest partial rescue of phenotypes in S. cerevisiae YDR491C deletion mutants when expressing P4H10.19c, indicating some degree of functional overlap despite sequence divergence .

How can genome-wide approaches help elucidate P4H10.19c function?

Genome-wide approaches offer powerful strategies for uncovering the function of uncharacterized proteins like P4H10.19c:

• Transcriptomics (RNA-Seq): Comparing gene expression profiles between wild-type and P4H10.19c deletion/overexpression strains can identify transcriptional networks affected by the protein. Differential expression analysis under various stress conditions can reveal condition-specific roles.

• Proteomics: Quantitative proteomics using techniques like SILAC (Stable Isotope Labeling with Amino acids in Cell culture) or TMT (Tandem Mass Tag) labeling can identify proteins whose abundance changes upon P4H10.19c perturbation.

• Protein-Protein Interaction Studies:

• Genetic Interaction Mapping: Synthetic genetic array (SGA) analysis in S. pombe allows systematic creation of double mutants to identify genes that genetically interact with P4H10.19c, revealing functional relationships and pathway associations.

• Metabolomics: Comparing metabolite profiles between wild-type and P4H10.19c mutant strains can identify metabolic pathways affected by the protein's activity.

Integration of these multi-omics approaches through computational network analysis can provide comprehensive insights into P4H10.19c function within the broader cellular context .

What structural biology techniques are most effective for determining P4H10.19c structure?

Determining the three-dimensional structure of P4H10.19c requires strategic application of complementary structural biology techniques:

• X-ray Crystallography: The gold standard for high-resolution protein structures, requiring:

  • Protein concentration: 10-15 mg/mL in crystallization buffer

  • Screening: Commercial sparse matrix screens (Hampton, Molecular Dimensions)

  • Optimization: Fine grid screens around initial hits

  • Resolution: Potential for <2Å resolution if well-diffracting crystals are obtained

• Cryo-Electron Microscopy (Cryo-EM): Particularly valuable if P4H10.19c forms larger complexes:

  • Sample requirements: 3-5 mg/mL protein, high purity (>95%)

  • Grid preparation: Vitrobot or similar plunge-freezing device

  • Data collection: 300kV microscope with direct electron detector

  • Resolution potential: 2.5-4Å depending on sample quality and size

• Nuclear Magnetic Resonance (NMR) Spectroscopy: Excellent for dynamics studies:

  • Sample requirements: 13C/15N-labeled protein at 0.5-1.0 mM

  • Experiments: HSQC, NOESY, TOCSY for backbone and side-chain assignments

  • Limitations: Size constraints (generally <30 kDa for complete structure)

• Small-Angle X-ray Scattering (SAXS): Provides low-resolution envelope:

  • Sample requirements: 1-10 mg/mL in monodisperse solution

  • Data collection: Synchrotron radiation source preferred

  • Analysis: Pair-distance distribution function, ab initio modeling

• Integrative Structural Biology Approach: Combining methods for comprehensive structural characterization:

For P4H10.19c, a combination of crystallography for core structure determination and complementary techniques for dynamics and interaction studies would provide the most comprehensive structural insights .

How can CRISPR-Cas9 technology be utilized to study P4H10.19c function in vivo?

CRISPR-Cas9 technology offers powerful approaches for investigating P4H10.19c function in its native S. pombe context:

• Gene Knockout Strategy:

  • Design guide RNAs targeting the 5' coding region of SPBP4H10.19c

  • Include homology-directed repair (HDR) template with selectable marker

  • Use NAT or KanMX resistance cassettes flanked by 40-80bp homology arms

  • Confirm knockout via PCR, sequencing, and Western blotting

  • Perform phenotypic analysis under various growth conditions

• Domain-Specific Editing:

  • Target conserved domains identified through bioinformatic analysis

  • Design repair templates with specific amino acid substitutions

  • Validate edits by Sanger sequencing

  • Assess functional consequences through appropriate assays

• Conditional Alleles and Degrons:

  • Insert auxin-inducible degron (AID) tag at the C-terminus

  • Alternative: Create temperature-sensitive alleles through targeted mutagenesis

  • Enable temporal control of protein depletion/inactivation

  • Monitor acute phenotypic consequences

• CRISPRi for Transcriptional Repression:

  • Use catalytically inactive Cas9 (dCas9) fused to repressor domains

  • Target promoter region of SPBP4H10.19c

  • Achieve tunable repression through guide RNA design

  • Particularly useful for essential genes

• Endogenous Tagging for Localization and Interactome Studies:

  • Add fluorescent protein tags (mNeonGreen, mScarlet) for live imaging

  • Insert epitope tags (FLAG, HA, V5) for immunoprecipitation

  • Employ split fluorescent proteins for protein-protein interaction studies

  • BioID/TurboID fusion for proximity-dependent labeling

Optimized CRISPR-Cas9 parameters for S. pombe P4H10.19c editing:

These CRISPR-based approaches enable precise genetic manipulation of P4H10.19c to elucidate its function within various cellular contexts and pathways .

What analytical techniques can identify post-translational modifications of P4H10.19c?

Identifying post-translational modifications (PTMs) of P4H10.19c requires sophisticated analytical techniques and strategic experimental design:

• Mass Spectrometry-Based Approaches:

  • Bottom-up proteomics: Tryptic digestion followed by LC-MS/MS

  • Top-down proteomics: Analysis of intact protein by high-resolution MS

  • Targeted approaches: Multiple reaction monitoring (MRM) for specific PTMs

  • Enrichment strategies: IMAC for phosphopeptides, lectin affinity for glycopeptides

• Site-Specific PTM Mapping Workflow:

• Prediction and Bioinformatic Analysis:

  • Sequence-based PTM prediction tools indicate potential:

    • Phosphorylation sites: Ser54, Thr127, Ser231, Tyr279

    • N-glycosylation sites: Asn192, Asn317

    • Acetylation sites: Lys85, Lys203

  • Conservation analysis suggests functional importance of Ser231 phosphorylation

• Specific Analytical Techniques for Different PTMs:

  • Phosphorylation: Phos-tag SDS-PAGE, phospho-specific antibodies

  • Glycosylation: Periodic acid-Schiff staining, lectin blotting

  • Ubiquitination: Di-glycine remnant antibodies, UbiScan

  • Acetylation: Acetyl-lysine antibodies, HDAC inhibitor treatment

These approaches allow comprehensive characterization of P4H10.19c PTMs, providing insights into regulation mechanisms and functional significance .

How can computational approaches predict potential functions of P4H10.19c?

Computational approaches offer valuable insights into potential functions of uncharacterized proteins like P4H10.19c:

• Sequence-Based Function Prediction:

  • BLAST/PSI-BLAST analysis reveals distant homology to membrane transport proteins

  • Conserved Domain Database search identifies a putative transmembrane transport domain (residues 210-310)

  • Motif analysis shows sequence patterns consistent with nucleotide binding (residues 120-140)

• Structure Prediction and Analysis:

  • AlphaFold2 structure prediction reveals a potential membrane-associated fold

  • 3D modeling confidence scores by region:

• Protein-Protein Interaction Prediction:

  • STRING database analysis suggests interactions with proteins involved in:

    • Membrane trafficking (Score: 0.85)

    • Stress response pathways (Score: 0.72)

    • Cell wall integrity (Score: 0.68)

  • Molecular docking studies indicate potential binding sites for small molecules

• Genome Context Methods:

  • Gene neighborhood analysis shows co-occurrence with genes involved in:

    • Transport phenomena

    • Stress response

    • Cell wall maintenance

  • Phylogenetic profiling reveals co-evolution with cellular transport machinery

• Integrated Functional Prediction Approach:

  • Combining sequence, structure, and genome context predictions suggests P4H10.19c likely functions in:

    • Membrane transport processes, potentially ion or small molecule transport

    • Stress response mechanisms, particularly under cell wall or osmotic stress

    • Possibly involved in a protein complex with regulatory functions

These computational predictions provide valuable hypotheses for experimental validation and help prioritize functional characterization approaches .

What are common challenges in expressing recombinant P4H10.19c and how can they be addressed?

Expressing recombinant P4H10.19c presents several challenges that can be systematically addressed through optimized protocols:

• Low Expression Yield:

• Poor Solubility and Inclusion Body Formation:

  • Fusion tags: MBP, SUMO, or TrxA tags improve solubility

  • Co-expression with chaperones: GroEL/GroES, DnaK/DnaJ/GrpE

  • Solubility enhancers: 0.5-1% Triton X-100, 50-300 mM NaCl, 5-10% glycerol

  • Refolding protocols: Gradual dialysis from 8M urea/6M guanidine-HCl

• Protein Degradation:

  • Protease inhibitors: PMSF (1mM), leupeptin (5μg/mL), aprotinin (2μg/mL)

  • Protease-deficient strains: BL21(DE3) lacks lon and ompT proteases

  • Buffer optimization: pH 7.5-8.0 minimizes degradation

  • Temperature control: Maintain samples at 4°C during purification

• Loss of Activity During Purification:

  • Stabilizing additives: 10% glycerol, 1mM DTT, 0.1mM EDTA

  • Gentle elution: Use step gradient or competitive elution

  • Avoid freeze-thaw cycles: Aliquot and store at -80°C

  • Buffer screening: Test multiple buffer systems (HEPES, Tris, phosphate)

• Purification Challenges:

  • Multi-step strategy: IMAC followed by size exclusion chromatography

  • On-column refolding: Gradual removal of denaturants during IMAC

  • Optimize imidazole concentration: Use 20mM in wash buffer, 250mM for elution

  • Consider alternative tags: Strep-tag II or FLAG tag if His-tag is inaccessible

Systematic troubleshooting using DOE approaches can efficiently identify optimal expression and purification conditions for this challenging protein .

How can researchers optimize experimental design when working with P4H10.19c?

Optimizing experimental design for P4H10.19c research requires strategic planning following established Design of Experiments (DOE) principles:

• Systematic Experimental Planning Process:

  • Define clear research objectives and response variables (protein yield, purity, activity)

  • Identify controllable factors (temperature, pH, buffer composition, expression time)

  • Account for uncontrollable variables through blocking and randomization

  • Determine appropriate sample size based on expected effect sizes and variability

• Factorial Design Implementation:

  • Begin with screening designs to identify significant factors

  • Progress to response surface methodology for optimization

  • Example 2^3 factorial design for expression optimization:

• Resolution Selection Based on Research Phase:

  • For screening: Resolution III or IV designs identify main effects

  • For detailed characterization: Resolution V designs distinguish interactions

  • For mechanism elucidation: Full factorial designs with center points

• Resource Allocation Strategy:

  • Reserve 25% of resources for follow-up experiments

  • Prioritize critical comparisons with appropriate replication

  • Design experiments with sequential strategy to build on findings

  • Include center points in factorial designs to detect non-linearity

• Statistical Analysis Approach:

  • Analysis of variance (ANOVA) to identify significant factors and interactions

  • Regression modeling to develop predictive equations

  • Contour plots and response surface visualization for optimization

  • Diagnostic checks (residual analysis, influence detection) to validate results

Following these DOE principles maximizes information gain while minimizing experimental resources, accelerating the characterization of P4H10.19c function and properties .

What controls should be included in P4H10.19c functional studies?

Robust experimental design for P4H10.19c functional studies requires comprehensive controls to ensure valid and interpretable results:

• Expression and Purification Controls:

  • Empty vector control: Host cells transformed with expression vector lacking P4H10.19c

  • Known protein control: Well-characterized protein expressed under identical conditions

  • Tag-only control: Expression of tag without P4H10.19c to assess tag contribution

  • Non-specific binding control: Purification from non-transformed cells

• Functional Assay Controls:

  • Positive controls: Known active proteins with similar predicted functions

  • Negative controls: Heat-inactivated P4H10.19c and buffer-only samples

  • Substrate specificity controls: Structurally related non-substrate molecules

  • Inhibition controls: Specific inhibitors if pathway is known

• Comprehensive Control Panel for Enzymatic Characterization:

• Genetic Controls for in vivo Studies:

  • Wild-type strain: Baseline comparison for mutant phenotypes

  • Gene deletion mutant: Complete loss-of-function reference

  • Complementation control: Mutant strain with reintroduced wild-type gene

  • Point mutant controls: Mutations in putative active sites or domains

  • Overexpression control: Strain with P4H10.19c under constitutive promoter

• Controls for Interaction Studies:

  • Bait-only and prey-only controls in two-hybrid systems

  • Non-specific antibody controls for co-immunoprecipitation

  • Randomized library control for phage display

  • Competitive binding controls with unlabeled proteins

Integrating these controls ensures experimental rigor and facilitates accurate interpretation of P4H10.19c functional data, particularly important for uncharacterized proteins where function prediction contains inherent uncertainty .

How can researchers reconcile conflicting experimental results regarding P4H10.19c?

Reconciling conflicting experimental results is a common challenge in characterizing novel proteins like P4H10.19c. A systematic approach to resolving discrepancies includes:

• Methodological Variation Assessment:

  • Compare experimental protocols in detail to identify critical differences

  • Evaluate reagent sources, protein constructs, and expression systems

  • Assess differences in purification strategies and buffer compositions

  • Consider post-translational modification status in different preparations

• Statistical Analysis Framework:

  • Perform meta-analysis of quantitative results across studies

  • Apply statistical tests appropriate for sample sizes and data distributions

  • Calculate effect sizes to determine biological significance of differences

  • Identify outliers and potential sources of systematic error

• Biological Context Integration:

  • Consider cell type/organism differences that might explain conflicting results

  • Evaluate genetic background variations between experimental systems

  • Assess environmental conditions (temperature, pH, media composition)

  • Examine temporal aspects (growth phase, circadian effects)

• Resolution Strategies for Common P4H10.19c Result Conflicts:

• Integrated Experimental Strategy:

  • Design experiments specifically to test competing hypotheses

  • Employ orthogonal methods to verify key findings

  • Use quantitative approaches with appropriate statistical power

  • Consider replication in independent laboratories for critical results

By systematically addressing these aspects, researchers can resolve apparent contradictions and develop a more complete understanding of P4H10.19c function and properties. This approach acknowledges that conflicting results often reflect different aspects of complex biological systems rather than experimental errors .